Entomological surveillance of invasive Aedes mosquitoes in Mazandaran Province, northern Iran from 2014 to 2020

Mosquitoes are the most important vectors of serious infectious diseases in the world. Dengue, Zika, chikungunya and yellow fever are emerging and re-emerging infectious diseases, associated with the distribution of two key vectors i.e. Aedes aegypti and Aedes albopictus throughout the world including countries neighbouring Iran. Entomological surveillance was planned and performed monthly from May to December during 2014–2020 in selected counties of Mazandaran Province, North of Iran, by ovitrap, larval collection, hand catch and human baited trap. Overall, 4410 Aedes specimens including 2376 larvae (53.9%) and 2034 (46.1%) adults belonging to six species, namely Aedes vexans, Aedes geniculatus, Aedes caspius, Aedes echinus, Aedes pulcritarsis and Aedes flavescence were collected and morphologically identified. Over the seven years of surveillance, Ae. aegypti and Ae. albopictus were not found by any sampling method. Aedes vexans and Ae. geniculatus were the most abundant species, their populations peaked in October and November and was positively correlated with precipitation and relative humidity. Aedes flavescence was a new species record for the province. A flowchart for planning and implementation of invasive mosquito surveillance for provincial health authorities in the country is proposed. These surveillance efforts provide basic and timely information for the health system to act promptly on integrated and intensified surveillance and control programs should Ae. aegypti and Ae. albopictus detected in the province.


Aedes mosquitoes sampling
A total of 4410 Aedes specimens including 2376 larvae (53.9%) and 2034 (46.1%) adults belonging to 6 species, namely Ae. vexans, Ae. geniculatus, Ae. caspius, Ae. echinus, Ae. pulchitarsis and Ae. flavescence were collected from Mazandaran Province and identified using morphological characteristics. Among these, Ae. flavescence was a new record for Mazandaran Province. No specimens of Ae. aegypti and Ae. albopictus were found during the monitoring program from 2014 to 2020 (Table 1). Although the highest number of Aedes mosquitoes were caught in 2017 (22%), the maximum and minimum species diversity was recorded in 2015 (with 6 species richness) and 2014 (with 2 species richness), respectively. The highest number of Aedes specimens was collected by larval sampling method in the study area (Table 1). Interestingly, no specimens were collected by ovitraps and inspection in ships. (Fig. 2) Spatio-temporal distribution of the Aedes species during the study period. Means and standard deviations of Aedes populations were calculated based on year, month and counties in the province. The nonparametric Kruskal-Wallis test showed that the population abundance of Ae. vexans and Ae. geniculatus changed significantly by year, month, and county over the study period (Table 2). These species showed the most significant differences in 2017 (29. 16 Fig. 3). Aedes vexans was collected with the maximum mean abundance in October (19.68 ± 25.604) and Ae. geniculatus in December (18.60 ± 16.008), which was significantly different from other months ( Table 2 and Fig. 3). There was no significant difference between the population abundance of other Aedes species by year, month, and county during the monitoring period in the area ( Table 2).
Effects of climate variables on the abundance of Aedes species. The meteorological variables (mean rainfall, temperature and humidity) affected the population dynamics of Aedes species by month, year and county. Generally, the density of Ae. vexans and Ae. geniculatus was associated with the meteorological factors i.e. the highest mean humidity (81.35%), temperature (21.3 °C) and rainfall (180 mm) as shown in Fig. 3. Detailed relationship between the monthly meteorological factors and the density of these species during the study period is depicted in Fig. 5. Spearman correlation analysis of Aedes population abundance showed that The trend of monthly population variations of Ae. vexans and Ae. geniculatus from 2014-2020. Apart from 2014, in nearly all other years of the study period, Ae. vexans and Ae. geniculatus were collected from the study areas. Only in 2015, both Ae. vexans and Ae. geniculatus were collected from May to December. However, heterogeneities were observed in terms of the beginning and end of the monthly activity of these species. The highest activity peaks of these species were in autumn as recorded in October in 2015, 2017 and 2018, and November in 2019 and 2020. Aedes geniculatus had a different seasonal activity pattern than Ae. vexans during the sampling years. The population of this species showed some secondary peaks in the first half of the sampling years (Fig. 5).
Habitat Characteristics of Ae. vexans and Ae. geniculatus. As summarized in Table 4, the majority of Ae. vexans (87.77%) and Aedes geniculatus (98.9%) was observed in natural habitats. These Aedes species preferred to lay eggs in permanent and stagnant water in semi shady condition. Aedes vexans was found further in transparent waters (60.04%) with muddy floor (74.2%) and presence of plant out/surface and under water (61.78%), while Ae. geniculatus was observed in opaque waters without vegetation mainly in tree holes. Forest edge, Marsh, grassland were most preferred breeding sites of Ae. vexans in the province.  www.nature.com/scientificreports/ Adults of these species were more collected in forest sites as well as in the ports (Fig. 2). The forest sites were covered by dense and tall trees, meadows, shrubs and flowers. The cottages and other types of human dwellings in these sites were located at a distance of maximum 100 m apart with gable roofs and concrete, mud and wooden walls. The ports were covered (with grass and flowers and small shrubs.
The sampling sites also have administrative buildings, large sheds, buildings for employees to rest at a distance of approximately 1-50 m from each other with a gable roof and concrete, brick and iron walls. Figure 7 shows the spatial distribution of two species Ae. aegypti and Ae. albopictus in the past and present in Iran.

Discussion
The present study provides the results of a comprehensive entomological surveillance program to assess the presence of invasive Aedes species in Mazandaran Province during 2014-2020. First and foremost, Aedes aegypti and Ae. albopictus the main vectors of dengue, chikungunya, Zika and yellow fever were not found at the points of entry and high-risk sites in Mazandaran Province throughout the surveillance period by none of the collection methods including ovitraps. Probably, one reason could be that these species have not yet entered or established in the province. Historically though, Aedes aegypti was active in the southern regions of Iran from 1920 to 1951 [23][24][25] , it subsequently disappeared probably as a result of malaria eradication program which began Table 1. Numbers and percentage of Aedes specimens by different collection methods in Mazandaran Province, northern Iran during the entomological surveillance period, 2014-2020. The first two or three letters of the Counties names were used in this table. These include: Galugah County (Ga), Behshahr (Be), Neka (Ne), Sari (Sa), Ghaemshahr (Gh), Savadkooh (Sav), Fereydunkenar (Fe), Amol (Am), Mahmudabad (Ma), Noor (No), Noshahr (Nos)and Ramsar (Ra).  www.nature.com/scientificreports/ This was a top down surveillance strategy starting from the whole of the province down to the very exact counties with the potential points of entry (from outwards to inwards) to ascertain that Ae. aegypti and Ae. albopictus had not been established earlier in counties beyond the points of entry. Therefore, in the early years of the provincial surveillance program, the sampling process was carried out on a large scale throughout the province (16 counties). Subsequently, according to the national entomological surveillance protocol for Aedes aegypti and Ae. albopictus 11 , the surveillance program was limited to eight and then four counties that were considered high risk points of entry for the invasive Aedes. Specimens collected between May-December 2014 to 2020 provide baseline data on the relative abundance of Aedes species for the first time in the northern parts of Iran. The information builds up our understanding of the basic population dynamics, ecology and behaviour of local Aedes species.  Aedes vexans and Ae. geniculatus were collected more frequently in the study area, which is consistent with other studies in Iran [31][32][33] . To our Knowledge, there is limited and scattered data in the field of Aedes in Iran. In the studies conducted by Moradi Asal et al. 34 three Aedes larvae i.e. Ae caspius, Ae vexans and Ae. flavescens, and by Paksa  [46][47][48] . Since Ae. vexans is considered as the competent vector of West Nile virus, also considering its mammalophilic and ornithophilic bahavior, it can strengthen the role of "bridge vector" between birds and humans 49 . Considering its high abundance in the study area, laboratory virus surveillance in its populations is recommended. Although Ae. vexans was not found infected with WNV in Mazandaran Province, the virus was detected in Ae. caspius 50 , a species with the third rank in terms of abundance in the present study 51 . Therefore, it shows the circulation of the virus in mosquito populations in the northern parts of Iran, and risk of entry and spread of arbovirus diseases in the country 52 . Larvae of Ae. geniculatus were mainly observed in tree trunk cavities in northern part of Iran 33,36 . Similar to invasive Aedes, the species breeds in natural containers in woodland and man-made containers in the semiurban and semi-domestic environments and adults coexist with Ae. albopictus 33,53,54 . It is a Palearctic mosquito species, dispersed in North Africa, the Middle East, and all over Europe 55,56 , and documented for the first time from Mazandaran Province, north of Iran 57 , followed by Ardebil, Golestan and Guilan Provinces 58,59 . In Vitro studies showed that Ae. geniculatus can transmit yellow fever, eastern equine encephalitis 60 , Dirofilaria immitis, repens 61 and chikungunya virus 54 . Since the biology and ecology of Ae. geniculatus in Iran is poorly studied, further investigations are recommended.
Aedes vexans and Ae. geniculatus are known to be opportunistic feeders, day-active, exophilic mosquitoes that feed aggressively on birds, reptiles, humans and other mammals 56,62 . These species were collected with the highest mean frequency in Noshahr County (Fig. 3), a tourist destination and maritime trade hub in Mazandaran Province. Therefore, it poses a potential risk to human health in the area and highlights the importance of laboratory surveillance for arbovirus circulation in the region.
Aedes flavescence was found for the first time in the present study. The species was collected with low abundance in other parts of the world 15 . It was recorded for the first time in the form of larvae in West Azerbaijan in 1987 (Urmia city) 59 and recently documented as a new species in Ardabil Province 34 . Based on the results of these studies and considering the detection of the species in the current study, it seems that the species is distributed in the northern parts of Iran. It should be noted that there is not much information on ecological aspects of the species in Iran, warranting further studies.
There are many resemblances and differences between mosquitoes in choosing of breeding sites, knowing the type of preferred larval habitat of mosquito species is very important in planning control measures at the right place and time and reducing resources 63 . In our study, the most abundant species i.e. Ae. vexans and Ae. geniculatus were found more in natural habitats, including swamps and tree holes, respectively, than in artificial www.nature.com/scientificreports/ habitats. These species were mostly collected from permanent, semi-shaded habitats with mud beds. In agreement with the present study, Ae. geniculatus was found in natural habitats without vegetation, with muddy water, permanent and slow-flowing water, muddy bed in Golestan province, northeastern Iran 36 . Aedes vexans was also observed in natural habitats with vegetation, clean water, muddy bed, permanent and stagnant water in Hormozgan Province, southern Iran 64 . It was reported that these species lay their eggs in habitats exposed to sunlight 36,65 , whereas, in the present study they prefer habitats with semi shady conditions. Moosa-Kazemi et al. 37 reported that Ae. vexans tends to occupy habitats without vegetation in Kurdistan and Kermanshah Provinces, whereas our study and other studies 64,65 showed that this species lays eggs in habitats with vegetation. Unlike malaria vectors, there is not much data about the seasonal activity of Aedes species in Iran 66,67 . This is a preliminary report of monthly activities of the most abundant species of Aedes for the first time in northern Iran. In the present study, population fluctuations of Ae. vexans showed that its seasonal activity was mainly from May to December, while it was from June to December for Ae. geniculatus, both with the highest peaks in October and November. Wagner et al. 68 reported that these species were more active in Autumn and are seasondependent species in most cases. Aedes vexans was reported to be active from June to September in the Aras Valley, Turkey 69 , and from May to August in Fars Province, southern Iran 70 . The largest peak of the species was recorded in June 71 , August 72 and October 73 .
Climate and the environmental changes strongly affect the population dynamics of Aedes mosquitoes, alter the distribution, abundance, and longevity of mosquito species and consequently influences the epidemiology of vector-borne diseases worldwide 74 . Although comparison of monthly and yearly mean rainfall and the abundance of Ae. vexans and Ae. geniculatus populations in current study revealed some sort of correlation (in October and in Noshahr), this is not a consistent picture. Therefore, other variables such as physicochemical factors, vegetation, wind speed and predators may possibly be influencing the population variations of Aedes species in their habitats 75,76 . The complexity of the correlations between the rainfall and abundance of flood water mosquitoes e.g. Ae. vexans is shown by several studies 22,73,77,78 . Other meteorological factors including temperature and relative humidity may, on the other hand, play a role in defining the fluctuation of the populations of Ae. vexans and Ae. geniculatus 79 . These species were most frequently collected when and where the temperature and relative humidity are the highest. Also, no association was found between meteorological factors and other Aedes species, especially Ae. caspius, as the third abundant species in the present study. The population dynamics of Ae. caspius depends strongly on availability of areas flooded with brackish water during high tides 80 as it tolerates high salinities owing to its high capacity for osmotic regulation 81 . The species was found in a wide variety of coastal sites, both fresh and saline marshes, but is most abundant in salt marshes, hence, its populations being less rainfall dependent than other species 80 .
Conclusion: This is the first comprehensive entomological surveillance in line with the national program in northern Iran that provides the basic information on Aedes species in the study area, the results of which www.nature.com/scientificreports/ can be useful for health decision makers in planning and implementing vector control programs in the future. Aedes aegypti and Ae. albopictus were not detected during the 7-year entomological surveillance in Mazandaran Province, northern Iran. However, since these species have recently been detected in southern Iran, Mazandaran Province and the whole of the country are going to be invaded sooner or later by these invasive Aedes species. This plus the fact that the most abundant species in the present study (Ae. vexans and Ae. geniculatus) are vectors of some pathogens, necessitates re-enforcing the national entomological surveillance program especially at high-risk areas such as airports, seaports, ground crossings and major routes for early detection of arrival of invasive species followed by prompt prevention and control programs. Mosquito collection and identification. The sampling followed a top down strategy i.e. from the whole of the counties of the province down to the very exact counties with point of entry in representative fixed sampling sites. In other words, to begin with and in the first year of the study, entomological surveillance was performed monthly from May to December 2014 in all of the 16 counties of the province. In 2015, the surveillance was performed bimonthly only in 8 counties with potential points of entry. From 2016 to 2020, the bimonthly surveillance was limited to counties with higher potential points of entries (ports and airports) in 4 counties throughout the province, as was suggested by the Iran CDC surveillance guideline of invasive Aedes vectors 11 . The names of the counties subjected to entomological surveillance are given in Table 1.

Material and methods
During the course of the entomological surveillance, four different sampling methods including ovitraps, larval collection, hand catch and human baited trap were employed. Ovitraps (100), containing 10% hay infusion, were installed bimonthly indoors and outdoors in selected points at each county. A total of 5400 ovitraps were placed in 54 selected sites during seven years of monitoring across the province (Fig. 2). They were visited for the presence of eggs 72 h later. Larval surveys were also conducted in the preferredartificial and natural breeding sites in a radius of 500 m from each point of entry (Fig. 2). Based on the shapes and sizes of the breeding sites, 350 cc dipper was used for larger, and pipette and dropper were used for smaller breeding sites. Water-holding containers on the deck of ships were also inspected for mosquito larvae. Fourth instar larvae were preserved in a glass of lactophenol solution and transferred to the laboratory, mounted on microscope slides using Berlese medium and identified morphologically using the key for the mosquitoes of Iran 20 . A total of 868 selected larval sampling points were visited during the 7-year monitoring program throughout the province. Larval habitat characteristics such as habitat type (natural or artificial), habitat condition (permanent or temporary, standing or flowing), vegetation type (with or without vegetation), floor type, water condition (clear or turbid) and condition Sunlight (full or partial light or shaded) were recorded. Human baited collections were performed fortnightly near breeding sites in 47 selected stations (Fig. 2) from morning to sunset using two human baits and one collector. The mosquitoes were pinned and identified using appropriate keys 20 . In addition, adult mosquitoes were collected with aspirator (hand catch) from various parts of ships, including bedrooms, kitchens, etc., arriving from Russia (Astrakhan and Makhachkala ports), Kazakhstan (Aktau port), Turkmenistan (Turkmenbashi port) and Azerbaijan (Baku port) to the ports of the Mazandaran Province.
A collection form was designed and used to record all collection data in the present study. Standard forms were used to report the surveillance data to the Ministry of Health.
Collection of meteorological data. Meteorological information including temperature, rainfall and relative humidity were obtained from the Meteorological department of Mazandaran Province and used to analyze the relationship between these factors and the population fluctuation of Aedes species. www.nature.com/scientificreports/ Data analysis. Larvae and adults of each species were summed for statistical analyses and collectively reported in graphs and tables 2 . Samples were pooled for each habitat type regardless of collection date and reported as percentages 21 . Spearman's test was used to assess the correlation between the Aedes population and the meteorological variables. A regression analysis model was performed to elucidate the relationship between Aedes populations and climatic factors. The mean (± standard deviation [SD]) number of Aedes species caught by month, year and county were computed and then compared using Kruskal-Wallis test followed by post hoc tests at a significance level of 5%. Statistical software SPSS ver. 25 was used to perform all the analyses 22 .
In order to perform the spatial analyses, geographical coordinates were extracted from 159 sampling sites (54 ovitrap, 58 larval collection, and 47 adult collection stations) using GPS software, entered into Excel in KML format, converted to "shape file", and then transferred to ArcMap GIS10.8 software to prepare the spatial database of mosquitoes of Mazandaran Province, northern Iran.

Data availability
The datasets generated and/or analyzed during the current study are presented in the manuscript and also are available from the corresponding author on reasonable request.